Recombinant collagen type Ⅱ and preparation method and application thereof

Abstract

This invention provides a recombinant type II collagen, its preparation method, and its applications, relating to the fields of pharmaceutical and cosmetic technology. The recombinant type II collagen contains a first amino acid sequence, specifically: GVMGFPGPKGANGEPGKAGEK (SEQ ID NO.1). This sequence contains multiple amino acid residues, such as methionine and phenylalanine, which participate in electron or hydrogen atom transfer processes. The hydrogen atom of the first amino acid pairs with the lone pair electrons of DPPH free radicals, thereby scavenging DPPH free radicals and exhibiting good antioxidant effects. Furthermore, the first amino acid sequence is rich in glycine and proline, forming a flexible structure. Pro provides rigidity and steric hindrance to the peptide chain, while Gly provides flexibility, facilitating effective contact with free radicals and further endowing it with antioxidant activity. It can scavenge reactive oxygen species generated intracellularly and reduce cell apoptosis.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceuticals and cosmetics, and more specifically, to a recombinant type II collagen, its preparation method, and its application. Background Technology

[0002] Antioxidants are a crucial health issue of widespread concern in today's society. During normal metabolism, the human body continuously produces a large number of reactive oxygen species (ROS). When ROS production is excessive or the free radical scavenging system is impaired, the dynamic balance between free radical production and scavenging is disrupted, leading to abnormal accumulation of free radicals. This accumulation can trigger lipid peroxidation of cell membranes, damage to genetic material, and protein dysfunction, thereby accelerating skin aging and inducing chronic inflammatory responses and various degenerative diseases. Therefore, developing functional ingredients with effective antioxidant capabilities has become a core direction of continuous exploration in the pharmaceutical and cosmetic industries. Collagen, as a key structural protein accounting for approximately 30% of mammalian connective tissue and a core component of the extracellular matrix, plays an irreplaceable role in maintaining the structural integrity and physiological function of tissues and organs such as skin, ligaments, tendons, and internal organs. Type II collagen, in particular, is specifically distributed in tissues such as cartilage, vitreous humor, and intervertebral discs, and has a decisive influence on maintaining tissue elasticity, mechanical support, and extracellular matrix homeostasis. However, the practical application of natural type II collagen in pharmaceutical preparations and functional cosmetics faces significant obstacles due to the difficulty in obtaining animal-derived materials, large fluctuations in product purity, insufficient physicochemical stability, and potential immunogenicity risks. Summary of the Invention

[0003] This invention aims to address the problems of difficulty in obtaining natural type II collagen, large fluctuations in product purity, insufficient physicochemical stability, and potential immunogenicity risks.

[0004] To address the above problems, this invention provides a recombinant type II collagen, its preparation method, and its applications.

[0005] In a first aspect, the present invention provides a recombinant type II collagen protein comprising a first amino acid sequence, the specific sequence of which is as follows: GVMGFPGPKGANGEPGKAGEK (SEQ ID NO. 1).

[0006] Optionally, it consists of a first amino acid sequence repeated 19 times.

[0007] Secondly, the present invention provides a method for preparing recombinant type II collagen as described above, comprising the following steps: S1: Obtain the codon through the first amino acid sequence to obtain the target gene. Synthesize the target gene into an expression vector to obtain a plasmid. The specific sequence of the first amino acid sequence is as follows: GVMGFPGPKGANGEPGKAGEK(SEQ ID NO.1); S2: Transform the plasmid obtained in S1 into the expression host, screen for positive transformants, and obtain the expression strain; S3: The expression strain obtained in S2 is cultured to express the target protein and obtain recombinant type II collagen.

[0008] Optionally, the steps also include: S4: Separate and purify the recombinant type II collagen obtained in S3 to obtain purified recombinant type II collagen.

[0009] Optionally, in S4, recombinant type II collagen is separated and purified by one or more of the following methods: salting out, ultrafiltration, chromatographic chromatography, isoelectric point precipitation, and membrane separation.

[0010] Optionally, in S1, the first amino acid sequence is optimized by Pichia pastoris codon preference and then amplified by PCR or synthesized as a whole gene to obtain the target gene.

[0011] Optionally, in S1, the expression vector is the pPicZαA expression vector.

[0012] Optionally, in S2, the expression host is Pichia pastoris X. 33.

[0013] Optionally, S3 includes the following steps: S31: The expression strain obtained in S2 is inoculated into YDP culture medium for culture and activation to obtain the seed culture in the upper tank; S32: When the seed culture in the upper tank reaches an OD600 of 70 or higher, start adding glycerol aqueous solution. Stop adding when the OD600 reaches 110 to 120. Wait for the dissolved oxygen to rebound to below 100% before starting to add methanol for induction. Collect the bacterial cells by centrifugation. S33: Place the bacterial cells in a buffer solution, lyse the cells, centrifuge to collect the supernatant, express the target protein, and obtain recombinant type II collagen.

[0014] Thirdly, the present invention provides an application of the recombinant type II collagen as described above in the preparation of antioxidant compositions.

[0015] The beneficial effects of the recombinant type II collagen, its preparation method, and its application of the present invention are as follows: GVMGFPGPKGANGEPGKAGEK (SEQ ID NO.1) is the specific chemical structure of the first amino acid sequence. This sequence contains multiple amino acid residues, such as methionine (Met) and phenylalanine (Phe), which participate in the transfer of electrons or hydrogen atoms. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable free radical. The hydrogen atom of the first amino acid pairs with the lone pair electron of the DPPH free radical, thereby scavenging the DPPH free radical and exhibiting a good antioxidant effect. In addition, the first amino acid sequence is rich in glycine (Gly) and proline (Pro), forming a flexible structure. Pro makes the peptide chain rigid and sterically hindered, while Gly provides flexibility, making it easy to penetrate the cell membrane and enter the cytoplasm / mitochondria, which is conducive to effective contact with free radicals, further endowing it with antioxidant activity, and enabling it to scavenge reactive oxygen species (ROS) generated in cells and reduce cell apoptosis. The first amino acid sequence can be prepared by gene synthesis and other methods, overcoming the problems of limited sources, poor purity and stability, and strong immunogenicity of existing natural collagen. This recombinant protein has a well-defined sequence structure and can effectively scavenge free radicals and inhibit intracellular reactive oxygen species levels, thus showing application potential in the field of antioxidation and providing a foundation for the development of novel antioxidant functional ingredients. Attached Figure Description

[0016] Figure 1 This is a schematic flowchart of the preparation method of recombinant type II collagen according to an embodiment of the present invention; Figure 2 This is an SDS-PAGE electrophoresis image of the purified recombinant type II collagen solution from Example 1. Figure 3 The graph shows the in vitro DPPH free radical scavenging rate of different concentrations of recombinant type II collagen in Example 1. Figure 4 Example 2 shows the in vitro DPPH free radical scavenging rate curves of different concentrations of collagen for comparison. Figure 5 Intracellular ROS staining images of different groups in Example 3; Figure 6 The relative fluorescence images of intracellular ROS staining in different groups of cells in Example 3 are for effect purposes. Detailed Implementation

[0017] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention's description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first," "second," etc., mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0019] This invention provides a recombinant type II collagen, its preparation method, and its application.

[0020] An embodiment of the present invention provides a recombinant type II collagen, comprising a first amino acid sequence, the specific sequence of which is as follows: GVMGFPGPKGANGEPGKAGEK (SEQ ID NO. 1).

[0021] Specifically, recombinant type II collagen refers to collagen synthesized and expressed in host cells by introducing the gene sequence encoding type II collagen into a suitable expression host through genetic engineering. Compared with naturally sourced type II collagen, recombinant type II collagen has advantages such as stable source, high purity, small batch-to-batch variability, and low immunogenicity, making it easier for large-scale production and functional research.

[0022] The first amino acid sequence is the basic structural unit that makes up recombinant type II collagen. This sequence is designed to have a specific amino acid composition and arrangement to endow recombinant type II collagen with specific biological functions. By repeating or combining this basic sequence, recombinant collagen with specific molecular weight and structural characteristics can be constructed.

[0023] This recombinant type II collagen can be produced using a variety of expression systems. For example, it can be expressed in prokaryotic systems such as *E. coli*, or in eukaryotic systems such as yeast, insect cells, or mammalian cells. Different expression systems vary in expression efficiency, protein folding, and post-translational modifications, thus affecting the properties of the final product.

[0024] This recombinant type II collagen contains a first amino acid sequence. The introduction of this first amino acid sequence can be achieved through genetic engineering. Specifically, the gene encoding this first amino acid sequence can be synthesized or obtained through polymerase chain reaction (PCR) amplification, and then cloned into a suitable expression vector. By transferring the expression vector containing this gene into host cells, the recombinant protein can be expressed, thus resulting in a recombinant type II collagen containing this specific first amino acid sequence.

[0025] The specific sequence of the first amino acid is GVMGFPGPKGANGEPGKAGEK (SEQ ID NO.1). This specific sequence is the core functional unit of the recombinant type II collagen of this application. The precise composition and arrangement of this sequence are crucial for conferring specific biological activities to the recombinant type II collagen. For example, through gene synthesis technology, it can be ensured that the DNA fragment encoding this sequence is accurately constructed, thereby enabling the precise translation of the amino acid sequence GVMGFPGPKGANGEPGKAGEK (SEQ ID NO.1) during protein expression.

[0026] In this embodiment, GVMGFPGPKGANGEPGKAGEK (SEQ ID NO.1) is the specific chemical structure of the first amino acid sequence. This sequence contains multiple amino acid residues, such as methionine (Met) and phenylalanine (Phe), which participate in the transfer of electrons or hydrogen atoms. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable free radical. The hydrogen atom of the first amino acid pairs with the lone pair electron of the DPPH free radical, thereby scavenging the DPPH free radical and exhibiting a good antioxidant effect. In addition, the first amino acid sequence is rich in glycine (Gly) and proline (Pro), forming a flexible structure. Pro makes the peptide chain rigid and sterically hindered, while Gly provides flexibility, making it easy to penetrate the cell membrane and enter the cytoplasm / mitochondria, which is conducive to effective contact with free radicals, further endowing it with antioxidant activity, and enabling it to scavenge reactive oxygen species (ROS) generated in cells and reduce cell apoptosis. The first amino acid sequence can be prepared by gene synthesis and other methods, overcoming the problems of limited sources, poor purity and stability, and strong immunogenicity of existing natural collagen. This recombinant protein has a well-defined sequence structure and can effectively scavenge free radicals and inhibit intracellular reactive oxygen species levels, thus showing application potential in the field of antioxidation and providing a foundation for the development of novel antioxidant functional ingredients.

[0027] Optionally, it consists of a first amino acid sequence repeated 19 times.

[0028] Specifically, in the field of protein engineering, repeating specific amino acid sequence motifs is an effective strategy for constructing proteins with enhanced function and stability. This repeating structure amplifies the inherent properties of a single motif and forms a more macroscopic molecular structure. For the recombinant type II collagen involved in this application, the first amino acid sequence (SEQ ID NO.1) is repeated to mimic the repetitive structural characteristics of natural collagen, thereby constructing a protein molecule with a specific length and conformation. The 19 repetitions are an experimentally optimized and designed number of repetitions, designed to ensure that the recombinant type II collagen maintains the function of its basic structural units while obtaining sufficient molecular weight and structural stability to effectively exert its biological activity. This repeating structure can increase the overall flexibility of the protein and provide more opportunities for exposure of active sites, thereby improving its functional efficiency.

[0029] In this optional embodiment, by repeating the first amino acid sequence 19 times, the problem of insufficient or unstable antioxidant effect of a single amino acid sequence structure is effectively solved. Specifically, this repeating structure significantly increases the molecular length and overall stability of recombinant type II collagen, enabling it to form a more optimized flexible conformation. This flexible conformation facilitates key residues in the first amino acid sequence, such as methionine (Met) and phenylalanine (Phe), to participate more effectively in the transfer of electrons or hydrogen atoms, thereby enhancing its free radical scavenging ability. Simultaneously, regions rich in glycine (Gly) and proline (Pro), due to their inherent flexibility, are further strengthened in the repeating structure, promoting effective contact between recombinant type II collagen and reactive oxygen species. This precisely designed number of repetitions ensures the continuity and functionality of the recombinant type II collagen sequence, avoiding the insufficient activity or structural instability that may result from a single sequence, ultimately significantly enhancing the antioxidant potential of recombinant type II collagen.

[0030] like Figure 1 As shown, another embodiment of the present invention provides a method for preparing recombinant type II collagen as described above, comprising the following steps: S1: Obtain the codon through the first amino acid sequence to obtain the target gene. Synthesize the target gene into an expression vector to obtain a plasmid. The specific sequence of the first amino acid sequence is as follows: GVMGFPGPKGANGEPGKAGEK(SEQ ID NO.1); S2: Transform the plasmid obtained in S1 into the expression host, screen for positive transformants, and obtain the expression strain; S3: The expression strain obtained in S2 is cultured to express the target protein and obtain recombinant type II collagen.

[0031] Specifically, in this preparation method, step S1 aims to convert the amino acid sequence of the target protein into a nucleic acid sequence that can be recognized and expressed by the expression host, and integrate it into a suitable genetic vector. Obtaining the codon from the first amino acid sequence (SEQ ID NO. 1) typically involves optimization based on the codon preference of the target expression host to improve translation efficiency and protein yield. The target gene can be obtained in various ways, such as through chemical synthesis, amplification from genomic DNA or cDNA libraries via polymerase chain reaction (PCR), or construction using gene splicing techniques. The target gene is synthesized into the expression vector, typically using restriction endonuclease digestion and DNA ligase ligation to directionally insert the target gene between the promoter and terminator of the expression vector, thereby constructing a recombinant plasmid containing the target gene. The choice of expression vector depends on the intended expression system; for example, *E. coli* expression vectors, yeast expression vectors, baculovirus expression vectors, or mammalian cell expression vectors can be used.

[0032] Step S2 involves introducing the recombinant plasmid carrying the target gene into an organism capable of protein expression. Various methods exist for plasmid transformation into the expression host, with the specific choice depending on the type of host cell and experimental requirements. For example, for bacterial hosts, chemical transformation (such as calcium chloride combined with heat shock) or electroporation can be used; for yeast hosts, lithium acetate or electroporation can be used; and for mammalian cells, liposome transfection, calcium phosphate precipitation, or electroporation can be employed. After successful plasmid introduction, a selection mechanism is needed to identify and isolate host cells containing the target gene, i.e., positive transformants. Selection typically relies on selectable marker genes carried on the expression vector, such as antibiotic resistance genes or auxotrophic complementation genes. By culturing in a medium containing the appropriate selection agent, only host cells successfully infused with the plasmid can survive and grow, thus obtaining stable expression strains.

[0033] Step S3 aims to provide a suitable growth and expression environment for the obtained expression strain to promote the large-scale synthesis of the target recombinant protein. Cultivating the expression strain typically includes stages such as seed culture, fermentation culture, and induced expression. Seed culture aims to obtain a sufficient number of viable cells to lay the foundation for subsequent large-scale fermentation; fermentation culture is carried out under optimized conditions to maximize biomass; induced expression initiates the transcription and translation of the target gene under the action of specific inducers (such as isopropyl-β-D-thiogalactoside (IPTG), methanol, lactose, galactose, etc.), thereby synthesizing a large amount of recombinant type II collagen. Culture conditions, such as temperature, pH, dissolved oxygen level, and feeding strategy, need to be finely controlled according to the characteristics of the selected host and expression system. After expression, the crude product containing the target protein can be collected from the culture medium or cell lysate by preliminary separation methods such as centrifugation and filtration.

[0034] This embodiment provides a systematic, efficient, and stable method for preparing recombinant type II collagen. Specifically, step S1 optimizes the codons of the first amino acid sequence and constructs a recombinant plasmid, ensuring the accuracy and high-efficiency expression potential of the target gene, laying a solid foundation for subsequent protein production. Step S2, by transferring the plasmid into the expression host and screening for positive transformants, achieves precise selection of host cells with high expression capacity, effectively reducing the risk of ineffective transformation and ensuring the initial efficiency of protein expression. Step S3 optimizes culture conditions to induce the expression strain to produce the target protein, ensuring stable and high-yield expression of recombinant type II collagen, thereby overcoming the problems of limited natural collagen sources, insufficient purity, and large batch-to-batch variations in existing technologies. Overall, this preparation method optimizes the production process step by step from the gene level to protein expression, significantly improving the preparation efficiency and product consistency of recombinant type II collagen, and providing a reliable technical approach for obtaining recombinant type II collagen with specific antioxidant activity.

[0035] Optionally, the steps also include: S4: Separate and purify the recombinant type II collagen obtained in S3 to obtain purified recombinant type II collagen.

[0036] Specifically, step S4, "separating and purifying the recombinant type II collagen obtained in step S3," aims to effectively remove host cell proteins, nucleic acids, lipids, culture medium components, and other expression impurities from the crude product obtained in step S3, thereby significantly improving the purity of the target protein. Various methods can be used to achieve separation and purification. For example, gel filtration chromatography based on molecular size differences, ion exchange chromatography based on charge differences, hydrophobic interaction chromatography based on hydrophobicity differences, or affinity chromatography utilizing the specific binding ability of the target protein can be employed. Furthermore, preliminary or fine separation can be achieved through physicochemical methods such as precipitation (e.g., salting out, isoelectric point precipitation) or membrane separation techniques (e.g., ultrafiltration, microfiltration). These methods can be used individually or in combination to achieve the desired purification effect. "Obtaining purified recombinant type II collagen" refers to the recombinant type II collagen obtained after the above separation and purification steps having high purity and significantly reduced impurity content. Purified proteins typically have a more stable structure and stronger biological activity, meeting the stringent quality requirements of subsequent applications (such as the preparation of antioxidant compositions), ensuring their functionality and safety.

[0037] In this optional embodiment, host cell residues, expression impurities, and other contaminants in the recombinant type II collagen obtained in step S3 can be effectively removed. Specifically, the recombinant type II collagen obtained in S3 is isolated and purified, which can specifically remove non-target components from the crude protein, avoiding potential risks introduced by unpurified protein. Through this process, purified recombinant type II collagen is finally obtained, with significantly improved structural integrity, biological activity, and stability. This not only eliminates the interference of impurities on protein structure and activity, ensuring that the recombinant type II collagen has high purity and functionality in subsequent applications such as the preparation of antioxidant compositions, but also enhances its stability and effectiveness in the field of antioxidation, thereby better exerting its antioxidant potential and solving the problem of limited application due to insufficient protein purity.

[0038] Optionally, in S4, recombinant type II collagen is separated and purified by one or more of the following methods: salting out, ultrafiltration, chromatographic chromatography, isoelectric point precipitation, and membrane separation.

[0039] Specifically, salting out is a technique that utilizes the difference in solubility of proteins at different salt concentrations for separation. For example, by adding neutral salts such as ammonium sulfate or sodium chloride to the solution and gradually increasing the salt concentration, the target protein or impurities can be selectively precipitated. Alternatively, the pH value of the solution can be adjusted in synergy with the salt concentration to optimize the precipitation effect, achieving preliminary separation and impurity removal. Ultrafiltration, on the other hand, is a method that uses a semi-permeable membrane with a specific molecular weight cutoff to separate substances of different molecular weights in a solution under pressure difference. For example, ultrafiltration membranes with different molecular weight cutoffs (such as 10 kDa, 30 kDa, etc.) can be selected. A suitable membrane can be chosen based on the molecular weight of recombinant type II collagen for separation. Alternatively, tangential flow filtration (TFF) or dead-end filtration modes can be used to achieve effective separation and concentration of the target protein from small molecule impurities. Chromatographic chromatography is a technique that separates components in a mixture by utilizing the differences in their partition coefficients between the stationary and mobile phases. For example, ion exchange chromatography can separate proteins based on differences in their charges, such as cation or anion exchange; gel filtration chromatography (molecular sieve chromatography) can separate proteins based on their molecular size; affinity chromatography can separate proteins based on the specific binding between proteins and specific ligands; or hydrophobic interaction chromatography can separate proteins based on differences in their hydrophobicity. This allows for the precise separation and purification of recombinant type II collagen. Isoelectric point precipitation is a method that separates proteins by utilizing the characteristic that their total net charge is zero and their solubility is lowest at their isoelectric point (pI). For example, the pH of the solution can be precisely adjusted to the isoelectric point of recombinant type II collagen to induce selective precipitation, or temperature control or the addition of a small amount of organic solvent can be used to assist precipitation, removing insoluble impurities or performing preliminary separation. Membrane separation methods refer to techniques that use selectively permeable membranes to separate mixtures. For example, microfiltration can be used to remove particulate matter and bacteria, nanofiltration can be used to remove small molecule impurities and salts, or reverse osmosis can be used to remove ions and very small molecules to further purify the solution and ensure the quality of the final product.

[0040] In this optional embodiment, a multi-step, highly efficient purification process can be constructed. Specifically, salting out, as a preliminary separation method, effectively removes a large amount of impurity proteins, significantly reducing the load on subsequent purification steps; ultrafiltration effectively separates and concentrates recombinant type II collagen based on molecular weight differences, removing small molecule impurities; chromatographic chromatography provides high-resolution fine separation capabilities, further removing impurities with properties similar to the target protein, thereby significantly improving the purity of recombinant type II collagen; isoelectric point precipitation utilizes the unique properties of proteins for separation, effectively removing impurities with low solubility at specific pH values; and membrane separation methods can be used as fine filtration or sterilization steps to ensure the purity and sterility of the final product. The combination or selective use of these methods allows the purification process to specifically remove different types of impurities, thereby obtaining high-purity, high-stability recombinant type II collagen. This effectively solves the problems of low efficiency and insufficient purity of single purification methods, ensuring the quality of recombinant type II collagen and enabling it to be better applied in fields such as the preparation of antioxidant compositions, exerting its expected antioxidant activity.

[0041] Optionally, in S1, the first amino acid sequence is optimized by Pichia pastoris codon preference and then amplified by PCR or synthesized as a whole gene to obtain the target gene.

[0042] Specifically, codon preference optimization refers to adjusting the gene sequence encoding a target protein based on the codon usage frequency of a specific expression host (e.g., Pichia pastoris) to improve translation efficiency and protein expression levels. Pichia pastoris exhibits unique codon preferences; some codons are efficiently recognized and translated in Pichia pastoris, while others may lead to translation stagnation or inefficiency. Therefore, bioinformatics software tools, combined with a Pichia pastoris codon usage frequency table, can be used to replace low-frequency codons in the original nucleotide sequence corresponding to the first amino acid sequence (SEQ ID NO.1) with high-frequency synonymous codons. For example, automated optimization can be performed using specialized gene optimization software, or manual comparison and replacement can be performed based on published Pichia pastoris codon usage frequency data. Furthermore, the optimization process may also consider factors such as avoiding mRNA secondary structures, adjusting GC content, and eliminating repetitive sequences to further improve gene stability and translation efficiency. After obtaining the nucleotide sequence corresponding to the codon-optimized first amino acid sequence, the target gene can be obtained through PCR amplification or whole-genome synthesis. PCR amplification is an in vitro enzymatic synthesis technique for DNA fragments, used to rapidly and specifically replicate large quantities of target gene fragments from a small amount of template DNA. If a suitable template exists (e.g., an existing synthetic gene fragment or a plasmid containing the sequence), specific primers can be designed, and DNA polymerase can be used for cyclic amplification to efficiently obtain the desired gene. Whole-gene synthesis refers to the de novo construction of a complete gene fragment based on known nucleotide sequence information. Whole-gene synthesis is a direct and efficient approach when a suitable template for PCR amplification is unavailable, or when extensive modifications to the gene sequence are required (such as codon optimization as mentioned above). This method typically involves synthesizing a series of short oligonucleotide fragments, which are then assembled into a complete target gene through enzymatic ligation or assembly reactions (e.g., Gibson Assembly, Golden Gate Assembly, or Overlap Extension PCR).

[0043] In this optional embodiment, Pichia pastoris codon preference optimization is performed on the first amino acid sequence, and the target gene is obtained by combining PCR amplification or whole-genome synthesis technology. This application can significantly improve the expression efficiency and yield of recombinant type II collagen in the Pichia pastoris expression system. Codon optimization ensures a high degree of matching between the gene sequence and the Pichia pastoris translation mechanism, effectively avoiding translation stagnation and protein folding errors caused by codon mismatch, thereby improving the accuracy and speed of translation. At the same time, PCR amplification or whole-genome synthesis provides a flexible and reliable gene acquisition method, ensuring high-quality target gene acquisition whether it is efficient replication from existing templates or precise de novo construction. This optimization strategy solves the problems of low gene expression efficiency, difficult synthesis, or insufficient expression levels in the expression host at the gene level, laying a solid foundation for the successful expression and large-scale preparation of recombinant type II collagen, and thus ensuring the quality and stability of the final product.

[0044] Optionally, in S1, the expression vector is the pPicZαA expression vector.

[0045] Specifically, the pPicZαA expression vector is a highly efficient secretory expression vector specifically designed for the Pichia pastoris expression system. This vector typically contains a strongly inducible promoter, such as the AOX1 promoter, which drives high-level transcription of the target gene under specific induction conditions (such as methanol). Furthermore, the pPicZαA vector integrates an α-factor secretion signal peptide sequence, which guides the secretion of the expression product extracellularly, simplifying subsequent purification steps. To ensure transformant selection, the vector also carries a selection marker gene, such as the Zeocin resistance gene, ensuring that only host cells successfully transformed and integrated with the vector can grow in selective media. Besides the pPicZαA expression vector, those skilled in the art can choose other vectors suitable for the Pichia pastoris expression system, such as the pGAPZαA expression vector, which utilizes the constitutive GAP promoter for sustained expression without the need for an inducer. However, pPicZαA, due to its inducible strong promoter characteristics, has significant advantages when high-level, controllable expression of secreted proteins is required.

[0046] In this optional embodiment, the pPicZαA expression vector is used in step S1 of the method for preparing recombinant type II collagen, which fully utilizes its high compatibility and high-efficiency expression characteristics in the Pichia pastoris expression system. The strongly inducible promoter contained in the pPicZαA vector ensures that the target gene encoding recombinant type II collagen can be efficiently transcribed and translated, thereby significantly improving the expression level of the target protein. Simultaneously, its carried secretion signal peptide guides the secretion of recombinant type II collagen into the culture medium, which not only reduces the risk of intracellular protein aggregation but also effectively simplifies the subsequent isolation and purification process, improving the overall preparation efficiency and purification yield. This vector selection avoids the problem of low expression efficiency or instability caused by inappropriate expression vector selection, laying a solid foundation for the plasmid transfer to the expression host in step S2 and the culture of the expression strain and expression of the target protein in step S3, thus ensuring the stable and efficient preparation of recombinant type II collagen.

[0047] Optionally, in S2, the expression host is Pichia pastoris X. 33.

[0048] Specifically, Pichia pastoris X-33 is a methanol-nutritional yeast strain widely used for recombinant protein expression. As an expression host, it offers advantages such as rapid growth, high fermentation density, large expression levels, ease of genetic manipulation, and the ability to perform correct folding and post-translational modifications (e.g., glycosylation) of eukaryotic proteins. These characteristics make it particularly suitable for expressing complex proteins derived from eukaryotes, ensuring that the expressed proteins possess the correct spatial structure and biological activity. Furthermore, the culture conditions for Pichia pastoris X-33 are relatively simple, facilitating large-scale industrial production and thus reducing production costs.

[0049] In this optional embodiment, using Pichia pastoris X-33 as the expression host effectively solves problems such as insufficient expression levels, protein folding errors, or loss of function that may occur during the expression of recombinant type II collagen. The efficient expression system of Pichia pastoris X-33 ensures that recombinant type II collagen can be obtained in high yields, while its eukaryotic expression environment facilitates correct protein folding and necessary post-translational modifications, thereby ensuring that the prepared recombinant type II collagen possesses the expected structure and antioxidant activity. This choice not only improves the preparation efficiency of recombinant type II collagen but also ensures its functional integrity, enabling it to effectively exert its antioxidant effects.

[0050] Optionally, S3 includes the following steps: S31: The expression strain obtained in S2 is inoculated into YDP culture medium for culture and activation to obtain the seed culture in the upper tank; S32: When the seed culture in the upper tank reaches an OD600 of 70 or higher, start adding glycerol aqueous solution. Stop adding when the OD600 reaches 110 to 120. Wait for the dissolved oxygen to rebound to below 100% before starting to add methanol for induction. Collect the bacterial cells by centrifugation. S33: Place the bacterial cells in a buffer solution, lyse the cells, centrifuge to collect the supernatant, express the target protein, and obtain recombinant type II collagen.

[0051] Specifically, in step S31, the expression strain obtained in S2 is inoculated into YDP medium for culture and activation, aiming to provide a sufficient number of physiologically active cells for subsequent large-scale fermentation. YDP medium is a commonly used yeast enrichment medium, typically containing nutrients such as yeast extract, peptone, and glucose, which can support rapid growth of the strain. Besides YDP medium, other nutrient-rich media suitable for yeast growth can also be used, such as BMGY (Buffered Minimal Glycerol Yeast Medium) or BMMY (Buffered Minimal Methanol Yeast Medium) for the initial growth stage, or customized composite media based on the nutritional requirements of specific yeast strains. The culture and activation process typically involves shaking culture at a suitable temperature (e.g., 28-30°C) and shaking speed to ensure full activation of the cells and reaching the logarithmic growth phase, providing a high-quality seed culture for subsequent fermenter inoculation.

[0052] In step S32, when the seed culture in the upper tank reaches an OD600 of 70 or higher, glycerol aqueous solution is added. Addition is stopped when the OD600 reaches 110-120, and the dissolved oxygen recovers to below 100%. Methanol is then added for induction, and the cells are collected by centrifugation. This step details the key control strategies for high-density fermentation and induced expression. The OD600 value is a commonly used indicator of cell density. Adding glycerol aqueous solution when the OD600 reaches 70 or higher aims to promote further accumulation of cell biomass, laying a solid cellular foundation for subsequent protein expression. The glycerol aqueous solution addition rate can be constant or gradient-added according to the cell growth to maintain optimal cell growth. Stopping glycerol addition when the OD600 reaches 110-120 ensures high-density cell accumulation. Subsequently, once dissolved oxygen rebounds to below 100%, indicating that the bacteria have largely depleted the carbon source in the culture medium and their physiological state has changed, methanol is added for induction. This efficiently activates the methanol-inducible promoter, thereby achieving high-volume expression of the target protein. The methanol can be added at a constant flow rate or gradually increased to avoid methanol toxicity and optimize induction efficiency. After induction, the bacteria are collected by centrifugation to separate them from the fermentation broth, preparing for subsequent cell disruption and protein extraction. Besides centrifugation, cross-flow filtration (TFF) or flocculation sedimentation can also be used for bacterial collection.

[0053] In step S33, the bacterial cells are placed in a buffer solution, the cells are lysed, and the supernatant is collected by centrifugation to express the target protein, thus obtaining the recombinant type II collagen. This step aims to release and preliminarily purify the target protein from the collected bacterial cells. Choosing a suitable buffer solution is crucial, such as phosphate buffer, Tris-HCl buffer, or acetate buffer. The pH and ionic strength need to be optimized according to the characteristics of the recombinant type II collagen, and protease inhibitors, reducing agents (such as DTT), or chelating agents (such as EDTA) may be added to prevent protein degradation or aggregation. Cell disruption methods can be mechanical, such as high-pressure homogenization, bead milling, or ultrasonic disruption, or non-mechanical, such as enzymatic digestion (e.g., Zymolyase for yeast) or chemical lysis (e.g., using detergents). The specific choice depends on the bacterial cell type, the protein's intracellular location, and the scale of the process. After cell disruption, the supernatant is collected by centrifugation to separate the soluble target protein from cell debris and insoluble substances, thereby preliminarily obtaining a crude extract containing recombinant type II collagen.

[0054] In this optional embodiment, by precisely controlling the expression process of recombinant type II collagen, the problems of low expression efficiency, insufficient protein yield, or unstable quality caused by the lack of specific culture parameters and induction conditions in traditional fermentation processes are solved. Step S31 ensures the health and viability of the starting strain, laying the foundation for high-density fermentation; Step S32 maximizes cell biomass and the expression efficiency of the target protein by precisely controlling the timing and conditions of glycerol supplementation and methanol induction, significantly improving the yield of recombinant type II collagen; Step S33 provides an efficient method for cell disruption and preliminary protein recovery, ensuring the effective acquisition of the target protein. These optimization measures work synergistically to significantly improve the reliability, stability, and economy of recombinant type II collagen production, providing high-quality raw materials for subsequent separation, purification, and application.

[0055] Another embodiment of the present invention provides the application of recombinant type II collagen as described above in the preparation of antioxidant compositions.

[0056] Specifically, an antioxidant composition refers to a formulation of substances capable of scavenging or inhibiting the generation of reactive oxygen species (ROS), thereby reducing or preventing damage to organisms or materials caused by oxidative stress. This antioxidant composition can be used as an active ingredient in cosmetics, such as creams, serums, and masks for anti-aging and skin repair; it can also be used as an effective ingredient in pharmaceutical products, such as oral preparations and topical ointments for treating or preventing diseases related to oxidative stress; or it can be used as a functional factor in functional foods or health products, such as beverages and tablets for enhancing the body's antioxidant capacity. During preparation, the aforementioned recombinant type II collagen can be effectively integrated into the target composition to enable it to exert the expected antioxidant activity. For example, the aforementioned recombinant type II collagen can be directly dissolved or dispersed in a suitable solvent system, and then mixed with other excipients (such as emulsifiers, stabilizers, thickeners, preservatives, etc.), and prepared into liquid formulations, emulsions, gels, or ointments through conventional formulation processes (such as stirring, emulsification, homogenization, filtration, etc.). In addition, to improve the stability and bioavailability of the recombinant type II collagen, it can be processed by techniques such as microencapsulation, liposome encapsulation, or nanoparticle loading before being added to the composition.

[0057] In this embodiment, the recombinant type II collagen is applied to the preparation of antioxidant compositions, effectively addressing the gap in the antioxidant applications of this protein in the prior art, thereby significantly expanding its use in functional products. This recombinant type II collagen, particularly when composed of the first amino acid sequence repeated 19 times (GVMGFPGPKGANGEPGKAGEK (SEQ ID NO.1)), possesses a specific amino acid sequence structure that endows it with significant antioxidant activity. Specifically, the methionine (Met) and phenylalanine (Phe) residues that may be present in this sequence can participate in the transfer of electrons or hydrogen atoms, thereby effectively scavenging free radicals. Furthermore, the flexible structure rich in glycine (Gly) and proline (Pro) facilitates its full contact with free radicals, enhancing its free radical scavenging ability. Through this application, the recombinant type II collagen not only exhibits free radical scavenging ability in the DPPH system but also shows a significant inhibitory effect on hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species levels, providing a new source of active ingredients with clear antioxidant functions for the pharmaceutical and cosmetic fields. This application fills a gap in the field of antioxidant recombinant type II collagen, enhancing its value and practicality as a functional biomaterial.

[0058] The present invention will be further described below with reference to specific embodiments.

[0059] Example 1: Preparation and purification of recombinant type II collagen.

[0060] 1. The amino acid sequence SEQ ID NO.1 of recombinant type II collagen was optimized according to the codons of the yeast expression system to obtain the target gene sequence. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was ligated into the pPicZαA plasmid to obtain the pPicZαA-Ⅱ001 plasmid.

[0061] 2. pPicZαA-Ⅱ001 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Transformants were screened using bleomycin resistance as a selection marker to obtain the yeast expression strain.

[0062] 3. Select a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubate at 30℃ and 200 rpm for 48 h for activation; A 1% inoculum was transferred to a 500ml Erlenmeyer flask (containing 200ml of YPD culture medium) and cultured at 30℃ and 200rpm for 24 hours as seed for the next fermentation. 3L of BSM culture medium was prepared and added to a 5L fermenter, sterilized at 121℃ for 20 minutes, and after cooling to 30℃, the pH was adjusted to 5.0. The medium was then added to the fermenter via flame inoculation. When the OD600 reached 70, 50% glycerol was added. Addition was stopped when the OD600 reached approximately 120. Once dissolved oxygen rebounded to 100%, methanol was added for induction. During induction, dissolved oxygen was controlled to be no lower than 30%, and the pH to be approximately 5.0. Induction was performed for 40 hours. Fermentation was then terminated. The culture medium was centrifuged at 12000rpm for 2 minutes; the supernatant was the crude recombinant type II collagen.

[0063] 4. The collected fermentation broth of the engineered bacteria was separated from the bacterial cells using a Thermo Fisher Scientific benchtop centrifuge, and the supernatant was collected. Based on the characteristics of this protein, a buffer solution was prepared: 20 mM / L potassium phosphate buffer (Solution A, pH 6.0), with 20 mM / L potassium phosphate buffer + 1 mol / L NaCl (Solution B, pH 6.0) as the elution buffer. After adjusting the pH of the collected supernatant, it was filtered and loaded onto a hydrophobic cation exchange chromatography column. Before loading, the column was equilibrated with Solution A. After loading, impurities were washed with 20% Solution B, and finally, elution was performed with Solution B. The eluted protein was the target protein, which is the purified recombinant type II collagen solution. Then, it was pre-frozen at -20°C for 4 hours, and then transferred to a vacuum freeze dryer for lyophilization. After 48 hours, the lyophilized protein was collected, which was the target protein, recombinant type II collagen. The recombinant type II collagen was measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the SDS-PAGE electrophoresis image was obtained, as shown below. Figure 2 As shown.

[0064] Example 1: The in vitro antioxidant properties of the recombinant type II collagen prepared in Example 1 were tested.

[0065] Weigh 5.0 mg of DPPH and dilute to 100 mL with anhydrous ethanol to prepare a 50 μg / mL DPPH ethanol solution. Prepare and use immediately, and store in the dark. Weigh 4 mg of recombinant type II collagen sample and add 2 mL of pure water to prepare a 2 mg / mL stock solution. Dilute this stock solution to prepare a series of concentration gradients of 1.0 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL as sample solutions. Use vitamin C solution as a positive control. Dissolve and dilute it with 95% ethanol to prepare a series of concentration gradients of 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL to verify the experimental system.

[0066] Referring to Table 1, sample wells (T), sample background wells (T0), DPPH wells (C), and solvent background wells (C0) were set up in the 96-well plate. Each reagent was added, and the plate was allowed to stand at room temperature for 5 min. After shaking the plate, the absorbance was measured at 517 nm.

[0067] Table 196: Liquid Addition Requirements for Orifice Plates

[0068] In vitro DPPH free radical scavenging rate curves of recombinant type II collagen at different concentrations were generated, such as... Figure 3 As shown, the scavenging rate of recombinant type II collagen against DPPH free radicals increased with increasing concentration in the range of 0.25 mg / mL to 2.0 mg / mL, with a scavenging rate exceeding 50% at 2.0 mg / mL, which is close to the results of the vitamin C group.

[0069] The DPPH free radical scavenging experiment used in this embodiment follows the method specified in the group standard T / SHRH 006-2018 "Cosmetics - Test Method for Free Radical (DPPH) Scavenging". However, since this embodiment uses recombinant type II collagen as the sample, considering protein stability and structural integrity, the above-mentioned optimizations were made to the concentration range, reaction time, and solvent buffer system. Comparative experiments demonstrate that the improved method can more accurately assess its scavenging ability while ensuring protein activity.

[0070] Comparative Example 1: Comparison of collagen preparation and purification.

[0071] 1. The second amino acid sequence GVMGFAGAVGANGEGAVAGEA (SEQ ID NO.2) was optimized according to the codons of the yeast expression system to obtain the comparative gene sequence. The obtained comparative gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was ligated into the pPicZαA plasmid to obtain the pPicZαA-Ⅱ002 plasmid.

[0072] 2. pPicZαA-Ⅱ002 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Transformants were screened using bleomycin resistance as a selection marker to obtain the yeast expression strain.

[0073] 3. Select a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubate at 30℃ and 200 rpm for 48 h for activation; A 1% inoculum was transferred to a 500ml Erlenmeyer flask (containing 200ml of YPD culture medium) and cultured at 30℃ and 200rpm for 24 hours as seed for the next fermentation. 3L of BSM culture medium was prepared and added to a 5L fermenter, sterilized at 121℃ for 20 minutes, and after cooling to 30℃, the pH was adjusted to 5.0. The medium was then added to the fermenter via flame inoculation. When the OD600 reached 70, 50% glycerol was added. Addition was stopped when the OD600 reached approximately 120. Once dissolved oxygen rebounded to 100%, methanol was added for induction. During induction, dissolved oxygen was controlled to be no lower than 30%, and the pH to be approximately 5.0. Induction was performed for 40 hours. Fermentation was then terminated. The culture medium was centrifuged at 12000rpm for 2 minutes; the supernatant was the crude contrast collagen.

[0074] 4. The collected fermentation broth of the engineered bacteria was separated from the bacterial cells using a Thermo Fisher Scientific benchtop centrifuge, and the supernatant was collected. Based on the characteristics of this protein, a buffer solution was prepared: 20 mM / L potassium phosphate buffer (Solution A, pH 6.0), with 20 mM / L potassium phosphate buffer + 1 mol / L NaCl (Solution B, pH 6.0) as the elution buffer. After adjusting the pH of the collected supernatant, it was filtered and loaded onto a hydrophobic cation exchange chromatography column. Before loading, the column was equilibrated with Solution A. After loading, impurities were washed with 20% Solution B, and finally, elution was performed with Solution B. The eluted protein was the control protein, which is the purified control collagen solution. It was then pre-frozen at -20°C for 4 hours, and then transferred to a vacuum freeze dryer for lyophilization. After 48 hours, the lyophilized protein was collected, which is the control collagen.

[0075] Example 2: The in vitro antioxidant properties of the comparative collagen prepared in Comparative Example 1 were tested.

[0076] Weigh 5.0 mg of DPPH and dilute to 100 mL with anhydrous ethanol to prepare a 50 μg / mL DPPH ethanol solution. Prepare and use immediately, and store in the dark. Weigh 4 mg of the control collagen sample and add 2 mL of pure water to prepare a 2 mg / mL stock solution. Dilute this stock solution to prepare a series of concentration gradients of 1.0 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.1 mg / mL as sample solutions. Use vitamin C solution as a positive control. Dissolve and dilute it with 95% ethanol to prepare a series of concentration gradients of 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL to verify the experimental system.

[0077] Referring to Table 2, sample wells (T), sample background wells (T0), DPPH wells (C), and solvent background wells (C0) were set up in the 96-well plate. Each reagent was added, and the plate was allowed to stand at room temperature for 5 min. After shaking the plate, the absorbance was measured at 517 nm.

[0078] Table 296: Liquid Addition Requirements for Orifice Plates

[0079] Construct comparative in vitro DPPH free radical scavenging rate curves for collagen at different concentrations, such as... Figure 4 As shown, the free radical scavenging ability of collagen is significantly reduced. Even with essentially the same peptide chain length and overall amino acid composition, replacing proline and lysine with hydrophobic amino acids, and only replacing key amino acids, can lead to a significant decrease in antioxidant capacity. This further demonstrates that the antioxidant effect of the first amino acid sequence in Example 1 originates from its specific amino acid composition and arrangement, exhibiting clear technical effects and good reproducibility.

[0080] Example 3: The recombinant type II collagen prepared in Example 1 was tested for its antioxidant properties at the cellular level. HaCaT cells were seeded in 24-well plates and divided into a normal group (DMEM medium, without collagen), a model control group (DMEM medium), and a sample group (DMEM medium). HaCaT cells in the model control and sample groups were induced with 400 μM H2O2 for 1 h after 24 h of culture. Each group was processed in triplicate. The sample groups were then treated with recombinant type II collagen solutions at concentrations of 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, respectively. Cells were cultured for 24 h, washed with PBS, stained with 10 μM DCFH-DA, and incubated at 37°C for 45 min. After washing with PBS, fluorescence images were captured. Figure 5 As shown in the figure. Parameters for quantitatively analyzing the expression levels of target proteins in each group were used by optical density measurement. The relative iodine values ​​(IOD values) of each group were plotted, as shown in the figure. Figure 6 As shown, # indicates a significant difference between the model group and the normal group, ###p<0.001, * indicates a significant difference compared with the model group, *p<0.05, **p<0.01, ***p<0.001. Figure 6 It is evident that recombinant type II collagen can significantly reduce intracellular ROS levels and has antioxidant effects.

[0081] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A recombinant type II collagen, characterized in that, It contains a first amino acid sequence, the specific sequence of which is as follows: GVMGFPGPKGANGEPGKAGEK (SEQ ID NO. 1).

2. The recombinant type II collagen according to claim 1, characterized in that, It consists of the first amino acid sequence repeated 19 times.

3. A method for preparing recombinant type II collagen as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Obtain the codon through the first amino acid sequence to obtain the target gene, synthesize the target gene into an expression vector to obtain a plasmid, wherein the specific sequence of the first amino acid sequence is as follows: GVMGFPGPKGANGEPGKAGEK(SEQ ID NO.1); S2: The plasmid obtained in S1 is transferred into the expression host, positive transformants are screened, and the expression strain is obtained; S3: Cultivate the expression strain obtained in S2 to express the target protein and obtain recombinant type II collagen.

4. The method for preparing recombinant type II collagen according to claim 3, characterized in that, It also includes the following steps: S4: Separate and purify the recombinant type II collagen obtained in S3 to obtain purified recombinant type II collagen.

5. The method for preparing recombinant type II collagen according to claim 4, characterized in that, In step S4, the recombinant type II collagen is separated and purified by one or more of the following methods: salting out, ultrafiltration, chromatographic chromatography, isoelectric point precipitation, and membrane separation.

6. The method for preparing recombinant type II collagen according to claim 3, characterized in that, In step S1, the first amino acid sequence is optimized by Pichia pastoris codon preference and then amplified by PCR or synthesized as a whole gene to obtain the target gene.

7. The method for preparing recombinant type II collagen according to claim 3, characterized in that, In S1, the expression vector is the pPicZαA expression vector.

8. The method for preparing recombinant type II collagen according to claim 3, characterized in that, In S2, the expression host is Pichia pastoris X.

33.

9. The method for preparing recombinant type II collagen according to claim 3, characterized in that, S3 includes the following steps: S31: The expression strain obtained in S2 is inoculated into YDP culture medium for culture and activation to obtain the seed culture in the upper tank; S32: When the seed culture in the upper tank reaches an OD600 of 70 or higher, start adding glycerol aqueous solution. Stop adding when the OD600 reaches 110 to 120. Wait for the dissolved oxygen to rebound to below 100% before starting to add methanol for induction. Collect the bacterial cells by centrifugation. S33: Place the bacterial cells in a buffer solution, lyse the cells, centrifuge to collect the supernatant, express the target protein, and obtain the recombinant type II collagen.

10. The use of the recombinant type II collagen as described in claim 1 or 2 in the preparation of antioxidant compositions.

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